Accumulating evidence indicates that nitric oxide (NO), through S-nitrosylation of Cys residues within multiple, functionally interrelated signaling elements, regulates agonist-induced desensitization and internalization of ?- adrenergic receptors (?-ARs). We have demonstrated that NO synthases and the endogenous S-nitrosothiol (SNO), S-nitrosoglutathione, preserve cardiac contractility and prevent down-regulation of ?-ARs during maintained agonist stimulation. These effects of NO/SNO on ?-AR signaling and trafficking, which can be recapitulated in cellular systems, appear to be mediated, in significant part, by S-nitrosylation of the G protein- coupled receptor (GPCR) kinase, GRK2. Additional components of the ?-AR system are also regulated by S- nitrosylation, which suggests a broad functional role for NO/SNO, exerted through targeted S-nitrosylation. In particular, we have recently identified the ?-arrestins (?arr1 and ?arr2) as targets of ?-AR-coupled S- nitrosylation by NO synthases (eNOS and nNOS). S-nitrosylation of ?arr2 by eNOS at a single critical site (Cys 410) regulates its protein-protein interactions with clathrin and adapter protein-2 (AP-2) in vitro and in vivo, thereby promoting agonist-mediated ?2-AR internalization. However, differential regulation of ?arr1 and ?arr2 by S-nitrosylation and the consequences of these modifications for cardiac function have not been explored. Our central hypothesis is that S-nitrosylation of the ?-arrestins will provide a basis for control by NO of ?-AR trafficking and signaling, with important ramifications in healthy and failing hearts. Moreover, we predict that the regulation of ?arr1- and ?arr2-specific interactomes by S-nitrosylation will provide a principal mechanism through which NO exerts its regulatory influence. We will carry out the following specific aims: 1. Elucidate the sites of agonist-dependent S-nitrosylation of the ?-arrestins by eNOS and nNOS in cells and tissues;2. Elucidate the consequences of ?-arrestin S-nitrosylation for ?-AR internalization and desensitization;3. Assess the consequences of ?-arrestin S-nitrosylation for ?-AR-dependent signaling;and 4. Assess the functional roles of ?-arrestin S-nitrosylation in the intact heart. Collectively, these studies should provide fundamental and novel insights into ?-AR regulation by NO in both healthy and failing hearts and may open a new area of research. PUBLIC HEALTH RELEVANCE: Heart failure is associated with both attenuated ?-adrenergic receptor (?-AR) signaling and decreased nitric oxide (NO) bioavailability. Because NO acts through S-nitrosylation to regulate both ?-AR densitization and downregulation, understanding the mechanisms of action of S-nitrosylation in ?-AR signaling and trafficking may improve understanding of ?-AR dysfunction and suggest novel therapeutic approaches to heart failure.